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Method and kit for synchronous fluorescence detection of hepatitis and HIV virus nucleic acid

A technology of HIV and kits, applied in the direction of microbial-based methods, biochemical equipment and methods, microbial determination/testing, etc., to achieve a wide range of optional effects

Inactive Publication Date: 2013-07-03
SHANGHAI XINGYAO MED TECH DEV CO LTD +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0020] The object of the present invention is to provide a kind of test kit that can extract the nucleic acid of HBV, HCV, HIV-1 virus synchronously, detect the nucleic acid of HBV, HCV, HIV-1 virus synchronously in real time, to overcome traditional protein detection and fluorescent quantitative PCR detection inherent At the same time, it makes up for many shortcomings of existing domestic and foreign similar products and even fatal flaws that cannot be solved in methodology

Method used

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  • Method and kit for synchronous fluorescence detection of hepatitis and HIV virus nucleic acid
  • Method and kit for synchronous fluorescence detection of hepatitis and HIV virus nucleic acid
  • Method and kit for synchronous fluorescence detection of hepatitis and HIV virus nucleic acid

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0075] Example 1 Preparation of biotin-modified capture probes and negative and positive controls 1 Construction of biotin-modified universal hybridization capture probes:

[0076] Entrust Sangon Bioengineering (Shanghai) Co., Ltd. to synthesize the following sequence:

[0077]

[0078] 5′-ACC ACC AAA TGC CCC TAT TTT TTT AGT ACC ACA AGG CCT TTCG AAA AAA CTA TGT TTC CCC TTG GTT CTC TC AAA AAA AAA AAA AAAAAAA AAA AAA-3′ and

[0079]

[0080] 5′-Biotin-Oligo(dT) 25 .

[0081] 2 Construction of positive control:

[0082] Entrust Sangon Bioengineering (Shanghai) Co., Ltd. to synthesize the following sequence:

[0083] HBV DNA positive control sequence:

[0084]

[0085] 5′-CGA CGA GGC AGG ACC CCT AGA AGA AAA AAA AAA AAA AAAAAAA AAA AAA-3′;

[0086] HCV RNA Positive Control Sequence:

[0087]

[0088] 5′-CUG CGG AAC CGG UGA GUA CAC AAA AAA AAA AAA AAA AAAAAAA AAA-3′;

[0089] HIV-1 RNA Positive Control Sequence:

[0090]

[0091] 5'-CCA UCA AUG AGG AAG CUG CAG AAU ...

Embodiment 2

[0096] Example 2 Specimen Processing: Magnetic Beads Method for Nucleic Acid Extraction and Reagent Preparation

[0097] 1 The composition of the sample processing reagent based on the principle of nucleic acid hybridization:

[0098] Lysis solution: 5M guanidine isothiocyanate, 50mM Tris, PH8.0, 10% NP-40, 10% Triton X-100, 6% SDS, 1μM capture probe, 5% polymer, etc.; for HBV, for HCV, for HIV-1.

[0099] Magnetic bead suspension: 1mg / mL streptavidin-coated magnetic beads, containing preservatives and dispersants such as Tween20;

[0100] Washing liquid W1: LiCl, Tris pH7.0, lithium dodecylsulfonate, preservatives and dispersants such as Tween20, pigments such as magenta or other fuels;

[0101] Washing liquid W2: LiCl, Tris pH7.0, preservatives and dispersants such as Tween20, etc.;

[0102] De-inhibitor: alcoholic solution containing protease and glycerol;

[0103] Eluent: Tris buffer prepared with DEPC-treated purified water, added with Tween 20 dispersant and EDTA,...

Embodiment 3

[0108] Example 3 Nucleic Acid Detection: Fluorescence Complementary Hybridization Probe Detection System Mode and Reagent Preparation

[0109] 1 Preparation of fluorescent complementary probe hybridization reaction solution:

[0110] 2×Annealing Buffer: Annealing buffer contains 30mM Tris·HCl (pH8.4), 50mM KCl, 2mM MgCl 2 , 5mM DTT and 1.5% formamide;

[0111] Fluorescent probe mixture: containing 5μM HBV, HCV, HIV-1 and internal standard fluorescent complementary probes, stabilizers and buffers. For each pair of complementary fluorescent-labeled probes, the amount of FAM probes is slightly more than that of non-FAM probes; and 4 pairs of probes are mixed with equal concentrations and equal volumes to make a probe mixture, and the concentration of each probe is about is 5 μM.

[0112] Reagent ratio and preparation: Mix 2×Annealing Buffer and fluorescent probe mixture according to the ratio of 25 μL: 5 μL, and add 20 μL nucleic acid sample.

[0113] 2×Annealing Buf...

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Abstract

Belonging to the technical field of nucleic acid diagnosis, the invention particularly relates to a method and a kit for synchronous extraction and synchronous detection of hepatitis and HIV virus by fluorescent complementary probe hybridization. With a magnetic bead as an automation medium, the method includes a technique for specific synchronous capture of HBV, HCV, and HIV-1 nucleic acids through biotin and oligonucleotide modified capture probes and a Taqman probe-based fluorescent complementary probe hybridization technique for real-time synchronous detections. The corresponding kit includes a disinhibition agent, a lysate, a magnetic bead suspension, a washing liquid, an eluent, an internal control, a 2 * Annealing Buffer, fluorescent probes, a positive control, and a negative control. The method and the kit provided in the invention have the characteristics of: no need of PCR reaction, single tube operation, closed detection, high degree of automation, synchronous extraction of various virus nucleic acids, and synchronous real-time detection, high sensitivity, good specificity and high precision, thus being suitable for large-scale blood screening, clinical examination and scientific research units.

Description

technical field [0001] The invention belongs to the technical field of diagnostic reagents—nucleic acid diagnosis, in particular to a highly sensitive, specific and suitable for synchronous extraction and synchronous real-time detection of hepatitis (including hepatitis B and hepatitis C) and AIDS in routine blood nucleic acid screening Methods and kits for viral nucleic acids. Background technique [0002] According to the statistics of the World Health Organization (WHO), more than half of the countries do not conduct thorough testing on the blood donated by blood donors, resulting in the spread of diseases such as AIDS, hepatitis, malaria and syphilis in many places. Of the 5.6 million HIV-infected people who increase globally every year, 5% to 10% of them are infected through blood transfusion or blood products. The proportion of virus carriers in African children and women is as high as 20% to 25%. Since the 1980s, countries such as France, Japan, Romania, the United ...

Claims

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Application Information

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IPC IPC(8): C12Q1/70C12Q1/68C12R1/93
Inventor 迟大利吴大治夏懿
Owner SHANGHAI XINGYAO MED TECH DEV CO LTD
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